Substrate specificity and regulation of the human SMG1-8-9 kinase complex

Langer, Lukas (2022): Substrate specificity and regulation of the human SMG1-8-9 kinase complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Quality control pathways are central to maintaining cellular homeostasis. On the mRNA level, nonsense-mediated mRNA decay (NMD) co-translationally detects and degrades transcripts with premature termination codons to prevent synthesis of truncated proteins, and regulates levels of physiological mRNAs. The phosphorylation of the RNA helicase UPF1 by the SMG1 kinase is considered a key step in this surveillance pathway: upon recognition of a premature translation termination event, it commits the targeted mRNA to degradation. SMG1 is known to recognize specific motifs within UPF1 for phosphorylation and to be regulated by both intra- as well as intermolecular interactions, but the details of both substrate recognition and activity regulation remain elusive. How the NMD factors SMG1 and UPF1 interface with the translation machinery is not understood. To address these questions, recombinant human SMG1 in complex with its binding partners SMG8 and SMG9 was purified from modified HEK293T suspension cells. In the first part of this study, the ~ 600 kDa kinase complex was reconstituted with a non-hydrolyzable ATP-analogue and a UPF1-derived peptide and subjected to single-particle cryo-electron microscopy (cryo-EM) analysis. The resulting 2.9Å reconstruction allowed to model a substrate peptide and a nucleotide analogue in the kinase active site. To further dissect the contribution of individual residues, modified SMG1 substrate peptides were assayed using mass spectrometry-based phosphorylation experiments. These results elucidated the molecular basis of specific phosphorylation site selection by SMG1 and provided insights into similarities and differences to related kinases, such as ATM and mTOR. Next, SMG1-8-9 was combined with a small molecule inhibitor specific for SMG1. The obtained structure provided insights into determinants of inhibitor specificity. Furthermore, the reconstruction revealed density attributed to a regulatory domain - the SMG1 insertion - blocking the substrate binding path within the kinase active site. Importantly, the reconstruction of a SMG1-9 complex calculated from the same data set did not show ordered SMG1 insertion density. By integration of the obtained structural data with crosslinking mass spectrometry, AI-based protein structure prediction and pull-down assays, this work found that SMG1 autoinhibition by its insertion domain is stabilized by the globular SMG8 C-terminal domain. These results shed light on the intricate regulatory network finetuning activity of this kinase complex. Additionally, leveraging in vitro translation methods, the reconstitution of four distinct eukaryotic translation termination complexes was established. Taken together, the work presented here describes the first structural model of a substratebound SMG1 kinase active site, revealing the molecular details of a central step of the NMD surveillance pathway. It provides insights into the regulation of the SMG1-8-9 kinase complex within NMD and offers a basis for rational design of small molecule compounds targeting SMG1. Finally, it paves the way to understanding NMD initiation by investigating the interaction between NMD factors and translation termination complexes.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 28. September 2022
• 1. Bericht­erstatter:in: Conti, Elena